This project focuses on the role that sub-cellular forces experienced at sites of adhesion between cells and their extracellular matrix play in regulating vascular smooth muscle cell organization and function. The adaptive process by which cells spatially resolve and respond to such localized forces is critical to adhesion remodeling, where cells reinforce specific adhesions while disassembling others, and is relevant to many mechanically-mediated disease processes such as the hypertension-induced hyperproliferative response of vascular smooth muscle cells that exacerbates vascular disease. Previous work in this area has shown that focal adhesions assemble in response to forces applied to them, but because adhesion to extracellular matrix also induces biochemical signals that trigger global cell contractility, it remains unclear whether forces applied to a specific adhesion result in any mechanical crosstalk with remaining adhesions distributed throughout the cell. This project will combine the expertise of two investigators to employ a new technique that simultaneously allows local mechanical stimulation of the adherent surface of a cell and spatially-resolved measurement of the local force fields generated throughout the cell in response to this stimulation. It is proposed that the relationship between local stimulation and global mechanical response is critical to the mechanical coordination within the cytoskeleton required for transduction of force into a meaningful response. The two investigators have recently developed a technique wherein deflections of an array of microfabricated posts report the cytoskeletal tension and local force fields generated by a cell attached to the array, and nanoengineered magnetic material embedded in individual posts is used to exerting tunable sub-cellular mechanical stresses to attached cells. Thus, cells can be locally perturbed at one post while the surrounding posts simultaneously measure the effects of this stimulation. Specific Aim 1 of the project will be to apply well- defined forces at the nanonewton level to individual adhesions in cells, and to measure the global response of a cell in terms of changes in contractility and the structure of focal adhesions. Specific Aim 2 will be to determine the role of adhesion signaling in the mechanical response to forces. Specific Aim 3 will be to investigate the role of the changes in cytoskeletal tension in regulating the focal adhesion response to forces. The experiments made possible by this novel technique will lead to new understanding of how mechanical stresses are transduced by cells into an adaptive, coordinated cytoskeletal response, and will open a pathway toward new insights into the mechanisms of mechanotransduction critical for inflammation, proliferation, and tissue development. PUBLIC HEALTH RELEVANCE: This research will apply recent advances in magnetic micro- and nanotechnology to study the response of vascular cells to force and mechanical stress. When cells in blood vessels are subject to abnormal stresses, such as occur in hypertensive arteriosclerosis, they display abnormal physical and biochemical responses that can further the progression of the disease. This research will provide new insight into these processes at the cellular level, and will have the potential to contribute significantly to the understanding of vascular disease.